We investigate the dependency of dynamic optical coherence tomography (DOCT) signal on wavelength and resolution in two systems. One is 840-nm spectral domain OCT, and the other is 1310-nm swept-source OCT. For wavelength dependency, we made the resolution roughly the same among two systems by computationally reducing the axial resolution of 840-nm system. For resolution dependency, we compared LIV images with and without reduction of the resolution. In addition, we performed numerical simulation by modeling the intracellular scatterer dynamics by a diffusion. Experiment and simulation suggested that LIV is highly affected by the wavelength, where shorter wavelength gives higher LIV.
We demonstrate longitudinal drug response imaging of tumor spheroids by integrating a spheroid cultivation chamber and the dynamic optical coherence tomography (DOCT) system. The cultivation chamber supports the spheroids with 5 % of CO2 and a temperature of 37 0C. In contrast to our previous cross-sectional time-course imaging method, this newly integrated system enabled longitudinal time-course imaging of a single sample, and hence enabled measuring large number of time-points of the same spheroid. It successfully revealed the temporal property of human breast cancer (MCF-7 cell-line) spheroid’s response to paclitaxel (PTX) and doxorubicin (DOX) with high-temporal-resolution.
We investigate wavelength and resolution dependency on dynamic optical coherence tomography (D-OCT) signal using two OCT systems. One is 840-nm spectral domain OCT. The other is 1310-nm swept-source OCT. We kept the resolution roughly the same between the two systems by computationally reducing the axial resolution of 840-nm system. In addition, we performed numerical simulation by modeling the intracellular scatterer dynamics by a diffusion. Experimental results showed that longer wavelengths correspond to lower D-OCT, while lower resolutions lead to slightly lower D-OCT. Simulation results confirm the wavelength dependency of D-OCT, with resolution variations having non-significant impact.
This presentation aims at classifying, mathematically modeling, and numerically simulating the intratissue activities for better understanding the dynamic optical coherence tomography (D-OCT) imaging. The intracellular motilities are classified into six types, and each type of motility is modeled by mono-direction translation, random ballistic, diffusion motions and their combination. The intracellular scatterer dynamic and D-OCT signals including logarithmic intensity variance (LIV) and complex variance were numerically simulated. These D-OCT signals increase within the velocity range of 4.5 to 270 nm/s and become almost constant for larger velocities. In addition, it was found that the shorter wavelength gives higher LIV and complex variances.
We demonstrated dynamic optical coherence tomography (D-OCT) to human skin in vivo by applying a sample fixation attachment and bulk motion correction algorithm to correct the motion artifacts. A D-OCT contrast of logarithmic intensity variance (LIV) was calculated. Without sample fixation attachment and the motion correction algorithm, the whole image area exhibited high LIV, and no meaningful structure was seen. The application of the motion correction methods revealed fine en face vessel structures, which cannot be seen in OCTA. The statistically significant motion artifact reduction capability of our motion correction method was also shown by t-test.
Dynamic optical coherence tomography (DOCT) is developed to evaluate the functional activities of wide spectrum of tissues. However, the relation between the DOCT signals and the intracellular motion is not fully identified yet. This unidentified relationship inhibits further dissemination of DOCT signals. In this study, we proposed a theoretical and numerical framework to understand DOCT. It includes the classification of intracellular motility, their mathematical modeling, and numerical simulation. We classified intracellular motilities into six types: active transport, passive transport, jiggling, floating of dissociated cells, migration, and flow. Then, the motilities were modeled by three physical models: flow, random ballistic and diffusion. The sample motion and it resulting time-sequential OCT images were numerically simulated. Two DOCT contrasts were computed from the OCT time-sequence: logarithmic intensity variance of OCT (LIV) and temporal variance of complex OCT signals (complex variance). We considered the random ballistic motions measured by two different probing wavelengths of 840nm and 1310nm. Tessellated pattern of low and high LIV was found in LIV images. The LIV and complex variance increase within the velocity range of 4.5 to 270nm/s, while it becomes almost constant for larger velocities. Additionally, we found that both LIV and complex variance are higher when shorter wavelength is considered. Using the proposed theoretical model, we can better understand the specific intracellular tissue activities that contribute to the high DOCT signal.
Dynamic optical coherence tomography (DOCT) is a method to visualize intratissue activities by analyzing the time sequence of OCT images. We previously established two DOCT contrasts, logarithmic intensity variance (LIV) and late OCT correlation decay speed (OCDSl), and applied them to several medical and pharmaceutical studies. However, these DOCT contrasts have two problems, which are a measurement time dependency of LIV and a difficulty of interpretation of OCDSl. Here we present a new DOCT algorithm which solves these two problems. The new method first computes several LIV values with multiple time window sizes. This LIV shows a monotonically increasing saturation curve. The saturation level and saturation speed, which are named authentic LIV (ALIV) and swiftness, are obtained by fitting the LIVs with a saturation function. Numerical simulation revealed that ALIV is sensitive to the occupancy of the dynamic scatterers over all dynamic and static scatterers, while swiftness is sensitive to the speed of the dynamic scatterers. According to the principle and experimental results using tumor spheroids, ALIV and swiftness are more quantitative and easier to interpret than our previous DOCT methods.
Dynamic optical coherence tomography (DOCT) is a label-free technique that visualizes tissue dynamics by analyzing a long-time sequence of OCT images. Although it was successful for in vitro and ex vivo imaging, it is still challenging for in vivo imaging because of the sample motion. We address this issue by developing hardware- and software-based motion suppression methods and demonstrating in vivo DOCT imaging of human skin. The hardware method is a sample fixation spacer. The software method is an image-registration based motion correction. We used logarithmic intensity variance (LIV) method to image the tissue dynamics. LIV was calculated from 32 sequential OCT frames taken within a 6.35s time window. The interframe time interval was 204.8ms and the entire DOCT volume was measured in 52.4s. Furthermore, we measured OCT angiography (OCTA) by standard raster scan with four frame repeats. To quantitively analyzing the improvement of proposed methods, three regions of interests (ROIs), each measuring 176μm (in depth) × 217μm (in lateral direction), was select from one B-scan to calculate the mean LIV. The improvement was assessed by paired t-test. The motion correction methods significantly reduced the high-LIV artifacts and revealed very fine capillary structures that had been buried by motion artefacts. The paired t-test results showed that the combination of fixation spacer and the software correction significantly reduced LIV artifacts (p=0.0052, 0.0137 and 0.0068 for three ROIs).
KEYWORDS: Optical coherence tomography, Point spread functions, Speckle, Signal processing, Signal analyzers, In vivo imaging, Artificial intelligence, Refractive index, Microscopy, Mathematical modeling
A new formulation of Optical Coherence Tomography (OCT) is presented. This formulation can represent a meaningful OCT image and speckles as two independent mathematical entities. In addition, by using the same formulation strategy, a new imaging modality to generate a spatially differential image, similar to differential interferometric contrast microscopy is demonstrated.
We demonstrate label-free dynamic optical coherence tomography-based evaluation of tumor spheroids' response to multiple anti-cancer drugs based on different mechanisms. The study involved dynamic imaging of human breast cancer (MCF-7) spheroids treated with tamoxifen citrate (TAM), Paclitaxel (PTX), and doxorubicin (DOX). In addition, fluorescence (FLUO) imaging was performed as a reference. The D-OCT imaging is performed using a custom-built OCT device with a repeated raster scan protocol. Two algorithms, including Logarithmic Intensity Variance (LIV) and late OCT Correlation Decay Speed (OCDSl) were used to visualize the tissue dynamics. The D-OCT visualized the drug type-dependent morphological and tissue-activity response patterns of the tumor spheroid. The different response patterns are clearly explained by the mechanisms of the drugs. The presented results suggest that D-OCT can be a useful tool for anti-cancer drug testing.
We demonstrate the label-free intra-tissue activity imaging of human induced pluripotent stem cell (hiPSC)-derived alveolar organoid including normal and fibrosis model with three-dimensional Dynamic Optical Coherence Tomography (D-OCT). The volumetric D-OCT data were obtained by a 840-nm spectral domain OCT. And the D-OCT images were obtained by “logarithmic-intensity-variance (LIV)” method, which is sensitive to the magnitude of tissue dynamics. Cystic structures with high-LIV border and mesh-like structures with low LIV were observed in all the organoids. They correspond to alveoli and fibroblast. The LIV images also visualized the abnormal repairing process of the alveolar epithelium.
Cancers is one of the most fatal diseases threatening the survival rate of humans. The recent advances in the cell culture methods allow cultivation of human-derived tumor cells as a 3-D culture, so-called tumor spheroid, which closely emulates the in vivo solid tumors. Hence, it can be used for anti-cancer drug testing, where the efficacy of anti-cancer drugs is evaluated by the drug-induced morphological and viability alterations of the spheroids. The morphological and tissue viability alterations can be evaluated by using staining histology, bright field microscopy, and fluorescence microscopy. However, these methods have several limitations. First, most of them use chemical labeling and/or tissue slicing, and hence, they are invasive. Second, they are 2-D methods. And hence, volumetric morphology, such as alteration of spheroid volume, cannot be evaluated. Third, their penetration-depth is limited to a few hundred microns. It prevents the imaging of thick tissues, such as spheroid. To overcome the above mentioned limitations, we developed a completely label-free 3-D dynamic optical coherence tomography (D-OCT) method for intracellular motility evaluation of tumor spheroid. To validate the utility of our D-OCT method, we organized several studies, including time-course and drug-response evaluations of human breast cancer (MCF-7 cell-line) spheroids. The 3-D morphological and cell viability alterations of MCF-7 spheroid during time-course and under the application of several anti-cancer drugs have been successfully visualized and quantified using D-OCT. The results suggest that D-OCT might be a useful for tumor spheroid-based drug testing and it might be a useful tool for precision medicine research.
Differential interference microscopy (DIC) is a method to obtain the refractive index distribution of a sample as contrast. It is suitable for biological cells, however, DIC can only obtain 2D images from thin samples. Therefore, we introduce a new imaging method, volumetric differential contrast (VDC) imaging using optical coherence tomography (OCT). This method enables getting 3D differential contrast of thick samples. VDC was designed based on the disperse scatterer model (DSM), a new theoretical model of OCT, and obtains differential contrast by complex numerical processing of OCT signal. DSM represents the sample as a spatially distributed refractive index with dispersed random scatterers, and OCT signal was formulated from this model. VDC uses two complex OCT signals, s1 and s2 at two laterally slightly distant positions, and the final image is defined as Im[s1 s2. This signal forms a spatially differential image of the product of the refractive index distribution and the scatterer density. According to the formulation, the size of the differentiation kernel, corresponding to the shear amount of DIC, is proportional to the defocus of the probe beam and the separation between s1 and s2. This method was validated by an in vitro spheroid sample and an in vivo zebrafish sample, measured by spectral domain OCT with a center wavelength of 830 nm. VDC images were obtained from refocused and defocused signals.
The zebrafish has shown to be an essential preclinical animal model, especially in the field of oncology. A non-invasive, high-resolution, and three-dimensional imaging modality is required to identify disease related changes in this model organism.
The presented polarization-sensitive Jones matrix optical coherence tomography (JM-OCT) prototype was utilized in three different studies, covering in vivo imaging of wildtype zebrafish, an adult postmortem tumor model and a longitudinal xenograft tumor zebrafish investigation.
This work highlights the potential of JM-OCT as a non-invasive, label-free, and three-dimensional imaging tool for preclinical cancer research based on zebrafish models.
We introduce volumetric differential contrast (VDC) imaging using optical coherence tomography (OCT). This method was designed based on a new theoretical model of OCT, the disperse scatterer model (DSM). VDC gives the differential image of “the product of the refractive index distribution and the scatterer density” through complex numerical processing of OCT signals.
The method was validated by in-vitro and in-vivo samples measured by spectral domain OCT. Differential contrast images with arbitrary shear amount and shear direction were obtained at arbitrary depth positions by a single measurement by numerically applying defocus by holographic signal processing after the signal acquisition.
Digital refocusing of optical coherence tomography (OCT) restores the spatial resolution degraded by defocusing. To apply this to imaging of biological tissue with point-scan Fourier domain OCT (FD-OCT), fast volume acquisition is needed to prevent motion disturbance. This study demonstrates the Lissajous-cycle-wise (LCW) digital refocusing algorithm applied to in vivo imaging at acquisition rates typical of FD-OCT. Blurring along and perpendicular to the scanning direction are compensated by inverse filtering and compositing A-lines of different scanning directions after motion correction, respectively. Ex vivo and in vivo biological sample experiments are applied for the proof of concept. The blurred images due to defocusing are sharpened by the LCW digital refocus algorithm.
KEYWORDS: Optical coherence tomography, In vitro testing, 3D modeling, Visualization, Tissues, Stereoscopy, Medical research, Lung, Drug development, Cancer
We demonstrate high-resolution (3.8-µm axial and 4.8-µm lateral) three-dimensional dynamic (D-) OCT imaging by 840-nm spectral domain OCT, and compare it with a low-resolution 1.3-µm D-OCT. The D-OCT images are obtained by logarithmic-intensity-variance method, which is sensitive to the magnitude of signal fluctuation.
Human-induced-pluripotent-stem-cell derived alveolar (lung) organoids and human breast cancer (MCF-7) spheroids were examined. The high-resolution D-OCT revealed the tessellation of high and low dynamics at the matured alveolar epithelium. It is also found that such matured alveolar epithelium exhibits ragged inner surface. For the spheroids, high-scattering spots with low dynamics were observed only in the high-resolution image.
We demonstrate OCT-based intracellular motility imaging method, so-called dynamic-OCT (D-OCT), and its application for tumor spheroid-based drug testing. The volumetric tomography is captured in 52.4 s using our custom-designed scanning protocol, which repeatedly capture 32 frames at each location in the tissue. Two algorithms including logarithmic intensity variance (LIV) and late OCT correlation decay speed (OCDSl) were used for tissue dynamics visualization. The utility of our proposed method is investigated for the comparison of three types of anti-cancer drugs applied to human breast cancer (MCF-7) spheroids. The drug type dependent alterations of cell morphology and viability have been successfully visualized.
In the biological and pharmaceutical research, there is a certain demand for label-free three-dimensional (3D) imaging of tissue function. Dynamic optical coherence tomography (OCT) has been demonstrated to fill this demand, but a very high-speed system is required for a volume acquisition of dynamic OCT. Here we demonstrate two new frame acquisition protocols which potentially enable the volumetric dynamic OCT by standard-speed OCT device with a reasonably short measurement time. One of the protocols takes multiple frames at a single location with a few hundred milliseconds interval. The other sequentially takes four frames at a single location, and then repeats this burst four-frame acquisition eight times with a few hundred milliseconds interval. These protocols were validated with in vitro and ex vivo samples. Both protocols gave reasonable dynamic OCT images.
We demonstrate volumetric phase contrast imaging by using optical coherence tomography (OCT). In general, the randomness of the scatterers’ distribution prohibits the volumetric measurement of a meaningful phase in a scattering mode. Our method uses complex numerical manipulation of an en-face complex OCT and gives a transversally differential phase image similar to a differential interference contrast microscope (DIC). Not like the DIC, our method can arbitrarily select the amount and direction of the shear after the OCT acquisition. In addition it provides DIC-like images at arbitrary depths. This method is validated by using a 840-nm spectral domain OCT system. A zebrafish sample is measured over a 1-mm × 1-mm transversal scanning range.
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